The erythropoietin receptor (EpoR), known for its role in the proliferation and differentiation of erythroid cells, is expressed in fetal liver, adult spleen, and adult bone marrow. We have also detected the EpoR in non-hematopoietic tissues such as fetal brain and vascular endothelial cells. To localize the embryonic expression of EpoR, we made transgenic mice with the hEpoR promoter driving the expression of beta-galactosidase. Unexpectedly, the pattern of expression seen in day 12 embryos with this construct localizes to the face, base of limbs and myotomes, similar to a pattern observed for embryonic myogenesis, driven by the MyoD transcription factor family. Examination of the 5'UTR of the hEpoR reveals three E-boxes (CANNTG), binding motifs for the MyoD family of transcription factors and other basic helix-loop-helix proteins (bHLH). The EpoR promoter activity in promyoblast C2C12 cells suggested that these E-box motifs may account for the similarity between embryonic EpoR transgene expression with that of Myf-5. The activity of the EpoR promoter/reporter gene construct is 60-fold over base line in C2C12 cells, but is markedly reduced when the E-box motifs are deleted. In contrast, there is no change in reporter gene activity in erythroid OCIM1 cells. During myogenesis the sequential expression of bHLH transcription factors Myf-5, MyoD, and myogenin compose a regulatory pathway that establishes myoblast identity and controls terminal differentiation. When C2C12 cells are grown in the presence of erythropoietin (Epo), the cells continue to proliferate and terminal differentiation is blocked. C2C12 cells that have terminally differentiated express MyoD and myogenin, while cells proliferating in the presence of Epo express Myf-5, low level of MyoD and no myogenin. These results suggest that Epo may promote a more primitive proliferative state by repressing the expression of the bHLH proteins essential for terminal differentiation. These studies will be extended to examine the role bHLH proteins, such as SCL/Tal-1, may play in the activation and/or response of EpoR during hematopoiesis/erythropoiesis.